Tissue acidosis (increased concentrations of extracellular protons or decreased pH) is associated with a number of painful physiological (e.g., cramps) and pathological (e.g., intermittent claudication, inflammation, ischemia, myocardial infarction). The extracellular pH may decrease by more than 2 log units during tissue acidosis (Reeh and Steen, 1996). The chemoreception of acid (protons) plays a critical role in the detection of nociceptive pH imbalances that occur in a number of conditions including camps, trauma, inflammation and hypoxia (Lindahl, 1974). Local tissue acidosis (increases in extracellular H+ concentration) arises from changes in the extracellular space during inflammatory or ischaemic conditions (Wall and Melzack, 1994). External acidification is a major factor in pain associated with inflammation, hematomas, cardiac or muscle ischemia, or cancer. Noxious chemical stimuli excite peripheral nerve endings of small diameter sensory neurons (nociceptors) in sensory ganglia (eg., dorsal root, nodose and trigeminal ganglia) and initiate signals that are perceived as pain. For instance, there is evidence that the sensation of pain parallels pH decreases (Steen et al., 1995). Prolonged intradermal infusion of low pH solutions can cause sensations that are similar to that felt during hyperalgesia, or chronic pain. Acid evoked currents in cardiac sensory neurons may mediate the sensation of myocardial ischemia (Benson et al., 1999). While decreasing the pH may cause a myriad of effects through a variety of mechanisms, the existence of ion channels that are directly gated (activated) by protons provides a means to pharmacologically manipulate specific pathways. A number of conductances in sensory and central neurons have been shown to be gated by low pH (Bevan and Yeats, 1991; Varming, 1999). Proton-gated cation channels with different pH sensitivities and kinetics were reported in sensory (trigeminal, cardiac and DRG) neurons (Benson et al., 1999; Bevan and Yeats, 1991; Kovalchuk et al., 1990; Krishtal and Pidoplichko, 1981), in neurons of the central nervous system (Grantyn and Lux, 1988; Ueno et al., 1992) and in oligodendrocytes (Sontheimer et al., 1989). The native proton-gated currents in DRG appear to vary among cells and include rapidly inactivating, non-inactivating and biphasic (both rapidly inactivating and subsequent non-inactivating) currents. A number of proton-gated channels have been cloned since 1996 and include the VR1 capsaicin-activated receptor (Tominaga et al., 1999) and a family of receptors that have homology to the nematode degenerin/mammalian amiloride-sensitive sodium channels (epithelial or brain Na channels; ENaC or BNaC). At least 4 of the latter have been shown to be expressed in sensory ganglia (see TABLE), suggesting a molecular correlate to the diversity of observed currents.
The cloned acid-sensing ion channels (ASICs) are structurally related to the Caenorhabditis elegans degenerins and mammalian epithelial sodium channels and are composed of 2 putative transmembrane domains and a large extracellular domain. The first member of this superfamily was the C. elegans deg-1 gene. A gain of function mutation of this gene (ie., the gene product was more active than wildtype) induced neuronal swellinng and degeneration (Chalfie and Wolinsky, 1990). This gene as well as a gene encoding a related protein MEC-4 (Driscoll and Chalfie, 1991) were called xe2x80x9cdegenerinsxe2x80x9d since they could mutate to toxic forms. There is a functional diversity of channels in this superfamily from H+ activated (ASICs), constitutively active (ENaC, Epithelial Na channel), peptide-gated (FMRF-receptor) in the snail to possibly stretch activation (degenerins of C. elegans). However, all of these family members appear to be sensitive to amiloride, are permeable to Na+, and are voltage insensitive (Waldmann and Lazdunski, 1998). With the exception of ASIC2b, all ASIC subunits form functional homomers when expressed in heterologous expression systems.
The kinetic properties of the homomeric channels vary. ASIC1 activates and desensitizes in the continued presence of acid and thus induces only a transient response (Waldmann et al., 1997). ASIC2 activates and inactivates more slowly than ASIC1 (Bassilana et al., 1997). DRASIC produces a biphasic response to extracellular acidification (Babinski et al., 1999; Waldmann et al., 1997). The sensitivity to acid depends on the subunit (see TABLE 1) and the pH producing the half-maximal current varies from about 6.5 to near 3.
Members of the degenerin superfamily form heteromers. This has been clearly shown with the ENaC family (Canessa, 1996; Fyfe and Canessa, 1998). ASIC subunits also appear to form heteromers. ASIC subunits may co-localize. At a tissue level, ASIC1A, ASIC2a and b, and ASIC3 mRNA are expressed the brain and ASIC1A and B, ASIC2b and ASIC3 mRNA are expressed in DRG (TABLE 2). For instance, ASIC2 and ASIC1 are co-expressed in almost all regions of mouse and human brain (Garcia Anoveros et al., 1997). Immunocytochemistry using polyclonal antisera against rat ASIC1 reveals ASIC1 protein in superficial dorsal horn, DRG and spinal trigeminal nucleus and peripheral nerve fibers (Olson et al., 1998). Functional co-expression of multiple ASIC subunits reveal channels with properties that differ from either homomeric channel, particularly in terms of the pH0.5, relative ion permabilities, and kinetics (Bassilana et al., 1997). ASIC2b is capable of modifying the kinetic and permeability properties of ASIC2a and DRASIC (Lingueglia et al., 1997). Furthermore, co-expression of ASIC family members as well as hASIC3 and the structurally similar P2X2 (Seguela and Babinski, 1999) produce receptors with properties that differ from either homomer. Messenger RNA encoding the present invention is localized to regions that express other ASIC subunits and suggest that hBNaC4 likely forms heteromers as well.
Most members of the amiloride sensitive Na channel/degenerin family appear to be highly selective for Na over K (Bassilana et al., 1997; Chen et al., 1998; Waldmann et al., 1997; Waldmann et al., 1997). The sustained component of DRASIC shifts from being largely mediated by Na to being less so in the presence of MDEG2 (Lingueglia et al., 1997). Chimers of MDEG1 and MDEG2 revealed nine amino acids in the putative N terminal cytoplasmic region adjacent to the first transmembrane region (TM1) are critically important determinants of the ion selectivity of these channels. Ile19, Phe-20 and Thr-25 appear to be particularly important since mutations in these residues discriminated poorly between Na and K (became xe2x80x9cnon-selectivexe2x80x9d) (Coscoy et al., 1999). Some ASIC subunits are also permeable to Ca2+: ASIC1A (PNa/PCaxcx9c2.5 (Waldmann et al., 1997)). However, not all subunits appear to form channels that are appreciably permeable to Ca2+ ((Bassilana et al., 1997; Chen et al., 1998)).
Amiloride block varies among subunits from a KD of about 10 uM to incomplete block at 100 uM (TABLE 2). While it is generally accepted that amiloride blocks the fast transient ASIC-mediated currents, there is some controversy as to the effect of amiloride on the sustained component of DRASIC (ASIC3) (Babinski et al., 1999; Bassilana et al., 1997; Waldmann et al., 1997).
Mutations in the degenerin genes encoding channels homologous to the ASICs causes neurodegeneration in C. elegans (Garcia-Anoveros et al., 1995). During the degeneration process, cells accumulate whorls (concentric spheres of membrane) and vacuoles, swell to several times their original diameter and often die (Hall et al., 1997). Similar mutations in the extracellular loop or second hydrophobic domain MII when introduced into MDEG1 and ASIC1 produce constitutively currents and kills cultured cells expressing the channel (Bassilana et al., 1997; Waldmann et al., 1996).
In vivo, activation of proton-gated channels would lead to cellular depolarization and influx of Ca2+ either through the channel itself or through voltage-activated calcium channels. Accumulation of intracellular Ca2+ may lead to neurodegeneration (Ghosh and Greenberg, 1995; Kristian and Siesjoe, 1996; Leist and Nicotera, 1999; Mattson, 1998; Sattler and Tymianski, 1998). Although tissue acidosis is a well established feature of cerebral ischemia, the role of acidosis in ischemic neuropathology is not understood. Previously, it had been thought to play a protective role since protons inhibit NMDA receptors (Giffard et al., 1990; Tang et al., 1990; Vyklicky et al., 1990). However, acidosis in vivo exaggerates damage due to ischemia even at a time when NMDA receptors are inhibited (Li and Siesjo, 1997; Siesjo et al., 1993).
The present invention describes the cloning and function of a novel ASIC receptor family member, BNaC4. The present invention is 97% identical at the amino acid level to rat xe2x80x9cSPASICxe2x80x9d (Genbank AJ242554), and therefore is a human ortholog of the rat gene. Members of the ASIC family are believed to participate in nociception but it is likely that they play other roles as well, especially in the central nervous system.
DNA molecules encoding human brain sodium channel BNaC4 that is a member of the ASIC (acid sensing ion channel family) have been cloned and characterized. The biological and structural properties of the protein are disclosed, as is the amino acid and nucleotide sequence. The recombinant protein is useful to identify modulators of the receptor BNaC4. BNaC4 is believed to play a role in neuromodulation, neurotransmission, pain, ischemia and neurodegeneration underlying diseases including but not limited to Alzheimer""s disease, Huntington""s disease, amyotrophic lateral sclerosis, cerebellar ataxias and Parkinsonism. Modulators identified in the assay disclosed herein are useful as therapeutic agents, which are candidates for the treatment of ischemia, inflammatory conditions and for use as analgesics for intractable pain, complex regional pain syndromes, arthritis (e.g., rheumatoid and osteoarthritis), as well as ulcers, neurodegenerative diseases, asthma, chronic obstructive pulmonary disease, irritable bowel syndrome, and psoriasis. Uses include the treatment of central nervous system diseases, diseases of the intestinal tract, abnormal proliferation and cancer especially in the digestive system, and female gonads, ulcer, liver disease, control of viscera innervated by the dorsal root ganglia, or to diagnose or treat any disorder related to abnormal expression of the human BNaC4 polypeptides, among others. In another aspect, the invention relates to methods to identify agonists and antagonists using the materials provided by the invention, and treating conditions associated with human BNaC4 imbalance. The recombinant DNA molecules, and portions thereof, are useful for isolating homologues of the DNA molecules, identifying and isolating genomic equivalents of the DNA molecules, and identifying, detecting or isolating mutant forms of the DNA molecules.